Sao Paulo Med. J. vol.118 número3

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75 INTRODUCTION

DNA typing techniques are o ne o f the mo st

ad-vanced to o ls fo r human identificatio n.

1

_{During the last}

10 years, a great number o f metho ds fo r DNA typing have

been intro duced to fo rensic science, with co nsiderable

success and also with co nsiderable co ntro versy.

2-4

_The

success and validatio n o f a criminal investigatio n are very

clo sely related to the pro cess used fo r o btaining and

pre-serving bio lo gical evidence.

5

Genetic markers can be characterized in traces o f

bio lo gical fluids, such as blo o d,

6,7

_sperm,

8

_saliva,

9,10

na-sal secretio n,

11

_{fragmented skeletal remains and o ld}

bo nes.

12-15

In severely charred fire victims bo th auto lytic

changes as well as deleterio us effects o f heat will cause

degradatio n o f the DNA.

16

Po lymerase chain reactio n (PCR) pro cedures

per-mit reliable replicatio n o f tho usand o f co pies o f a

spe-cific DNA sequence,

in vitro

, and have been described and

impro ved in recent years,

17-19

allo wing the study o f small

amo unts o f DNA even when degraded. These pro cedures

are therefo re extremely useful in the analysis o f fo rensic

samples. Several

loci

are especially suitable fo r PCR

analy-sis. DNA analysis has been used with success in the

iden-tificatio n o f carbo nized co rpses and victims o f large

ac-cidents.

16,20,21

_{The analysis do es, ho wever, require relatives}

o f crash victims to do nate blo o d fo r analysis. It is fo und

that the relatives are generally willing to co ntribute to

the identificatio n by giving a blo o d sample.

21

This paper describes the use o f PCR fo r genetic

characterizatio n o f o ne victim extensively burned by fire.

This identificatio n was po ssible by analyzing the DNA

Case R eport

REVISTA PAULISTA DE MEDICIN A

Post-morte m fore nsic ide ntity te sting:

application of PCR to the

ide ntification of fire victim

Department of Legal Medicine, Faculty of Medicine, Universidade de São Paulo, São Paulo, Brazil

a b s t r a c t

CO N TEX T:

DN A analysis has been used with success in the identifi-catio n o f carbo niz ed co rpses and victims o f larg e accidents.The analysis requires relatives o f crash victims to do nate blo o d fo r analy-sis. The relatives are g enerally willing co ntribute to the identificatio n by g iving a blo o d sample.

O BJECTIV E:

To describe the use o f the po lymerase chain reactio n (PCR) fo r g enetic characteriz atio n o f o ne victim extensively burned by fire.

DESIGN :

Case repo rt.

CASE REPO RT:

DN A was extracted fro m blo o d o f the cardiac cham-ber, and 1 5 different lo ci (D1 S8 0 , Apo B, D1 7 S3 0 , D3 S1 7 4 4 , D1 8 S8 4 9 , D1 2 S1 0 9 0 , FG A , D7 S8 2 0 , D1 S5 3 3 , D9 S3 0 4 , HUMCSF1 PO , HUMTPO X, HUMTHO 1 , amelo genin and HLA-DQ A1 ) were analyz ed using the PCR technique. Results fro m all lo ci typing o f the co rpse were then co mpared to that o f his alleg ed bio lo g ical parents, revealing a g enetic co mpatibility.

KEY W O RDS:

Fire victims. DN A typing . PCR. Human identificatio n. Paternity investig atio n.

•Jo sé Arnaldo So ares-Vieira • Ana Elisa Co rreia Billerbeck • Edna Sadayo Miaz ato Iwamura • Laís de Almeida Cardo so • Daniel Ro mero Muño z

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76 extracted fro m blo o d o f the cardiac chamber o f this

co rpse.

CASE REPORT

Scenario

During a recent po lice chase fo llo wing a

kidnap-ping, a car crashed and burned. An intensely burned

co rpse was remo ved fro m the car and sent fo r fo rensic

identificatio n. There were no fingerprints o r o ther ways

to identify the co rpse, but there was a suspicio n abo ut

its identity. To co nfirm this hypo thesis, the DNA o f the

alleged parents was co mpared to the DNA extracted fro m

the burned co rpse.

Procedures

DNA was extracted fro m blo o d o f the cardiac

cham-ber o f the carbo nized co rpse by the Kunkel metho d

22

_and

fro m 5 ml o f peripheral blo o d o btained fro m the alleged

bio lo gical parents o f the co rpse by the salting–o ut pro

-cedure.

23

_{The D1S80}

_locus

_{was studied using the D1S80}

Fo rensic DNA Amplificatio n Reagent Kit (Perkin Elmer,

USA), as reco mmended by the manufacturer. The

ampli-fied fragments were submitted to electro pho resis o n a

po lyacrylamide gel (GeneAmp Detectio n Gel – Perkin

Elmer) and visualized after silver staining. Allele

identi-ficatio n was achieved by co mpariso n o f the amplified

fragments to the allelic ladder included in the kit. The

study o f the HLA-DQA1

locus

and the analysis o f the

alleles were perfo rmed using the Amplitype HLADQA1 Fo

-rensic DNA Amplificatio n and Typing Kit (Perkin Elmer,

USA), as reco mmended by the manufacturer. The

ampli-ficatio n and analysis o f the D3S1744, D18S849, D12S1090,

FGA, D7S820, D1S533 and D9S304

loci

were assembled

using the co mpo nents o f the Multiplex I and Multiplex II

Kits (Lifeco des Co rp., USA). The CTT Multiplex Kit and

Amelo genin Kit (Pro mega Co rp., USA) was used fo r the

amplificatio n and study o f the HUMCSF1PO, HUMTPOX,

HUMTH01 and amelo genin

loci

. The Multiplex and

amelo genin kits were used as reco mmended by the

manufacturer. The D17S30

locus

was studied using

prim-ers, reactio ns and gel analysis as described by Ho rn.

24

Allele identificatio n was achieved by co mpariso n o f the

amplified fragments to the allelic ladder (Pro mega Co rp.,

USA). The Apo B

locus

was studied using primers,

reac-tio ns and gel analysis as described by Bo erwinkle.

25

Al-lele identificatio n was achieved by co mpariso n o f the

amplified fragments to the 100 bp and 123 bp ladder

(GIBCO-BRL, USA). The amplificatio n pro ducts o btained

fro m geno mic DNA extracted fro m the blo o d o f the co rpse

were co mpared to tho se generated fro m geno mic DNA

o btained fro m co rpse’s alleged bio lo gical parents.

The data o btained fro m the study o f the 15

loci

are

presented in Table.

DISCUSSION

The identificatio n o f remains fro m fire victims is

generally attempted by reco gnizing perso nal effects,

in-dividualizing marks (e.g. scars, tatto o s, signs o f kno wn

disease) and/o r dental reco rds.

Ho wever, due the effects o f heat and severe

lac-eratio n o f the bo dy, the identificatio n by usual fo rensic

means is no t always po ssible. In such cases DNA may be

o btained fro m crude tissue samples (abo ut 1 gram), co

l-lected during auto psy,

16

_{fro m so ft tissues within the nerve}

chamber o f unerupted wisdo m teeth

20

_{o r fro m muscles.}

21

A paternity test was perfo rmed and Mendelian

inherit-ance o f the alleles fo r these 15

loci

was o bserved.

The present study revealed that bio lo gical

mate-rial (blo o d) co llected fro m the cardiac chamber o f the

carbo nized co rpse, even with highly degraded DNA, co uld

be analyzed by the PCR technique and po sitively

iden-tify the victim.

Pro fessio nal wo rkers, like detectives, pro secuto rs,

lawyers, judges, do cto rs etc., who act in different fo

ren-sic areas related to the so lutio n o f crimes and human

identificatio n have lo ng had the need fo r an efficient

metho d capable o f po sitively identifying an individual.

Recent advances in mo lecular bio lo gy have significantly

enhanced the po tential fo r individualizatio n by pro

vid-ing a capability fo r typvid-ing DNA fro m vario us bio lo gical

materials. DNA analysis has been used successfully in a

number o f large accidents to asso ciate bo dy parts and

fo r the purpo ses o f identificatio n, by co mparing victims’

DNA pro files with tho se o f relatives. In this sense, mo

-lecular bio lo gy has beco me an impo rtant and

fundamen-tal to o l fo r perso nal investigatio n.

Table - Ge notype s ide ntifie d in the thre e DNA sample s

Lo cus AF AM CC RESULTS

HLA-DQ A1 4 , 4 1 .1 , 4 1 .1 , 4 N o exclusio n D1 7 S3 0 4 , 5 1 2 , 1 2 5 , 1 2 N o exclusio n

Apo B A , B B , C B , C N o exclusio n

D1 S8 0 2 0 , 3 1 2 1 , 2 4 2 0 , 2 4 N o exclusio n HUMCSF1 PO 9 , 1 0 1 0 , 1 1 9 , 1 1 N o exclusio n

HUMTPO X 8 , 9 7 , 8 7 , 8 N o exclusio n

Amelo g enin XY XX XY

-HUMTHO 1 9 , 1 0 6 , 8 8 , 9 N o exclusio n D3 S1 7 4 4 1 8 , 1 9 1 8 , 2 0 1 8 , 1 9 N o exclusio n D1 8 S8 4 9 1 7 , 1 8 9 , 9 9 , 1 7 N o exclusio n D1 2 S1 0 9 0 9 , 1 3 1 1 , 1 3 1 1 , 1 3 N o exclusio n FG A 2 5 , 2 6 2 4 , 2 4 2 4 , 2 6 N o exclusio n D7 S8 2 0 7 , 1 0 9 , 1 2 9 , 1 0 N o exclusio n D1 S5 3 3 1 3 , 1 4 1 5 , 1 5 1 3 , 1 5 N o exclusio n D9 S3 0 4 8 , 1 2 1 2 , 1 2 1 2 , 1 2 N o exclusio n

AF: alleg ed father o f carbo niz ed co rpse; AM: alleg ed mo ther o f car-bo niz ed co rpse; CC: carcar-bo niz ed co rpse.

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77

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r e s u m o

CO N TEX TO :

A análise do DN A tem sido utiliz ada co m sucesso na identificação de carbo niz ado s e de vítimas de g randes acidentes. A análise do DN A necessita de amo stras de sang ue do s familiares das vítimas co mo referência. O s familiares no rmalmente co ntribuem co m a identificação do ando uma amo stra de sang ue.

O BJETIV O :

Este trabalho descreve o uso da reação em cadeia de po limera se (PCR) pa ra c a ra c teriz a ç ã o g enétic a de uma vítima extensamente queimada pelo fo g o .

TIPO DE ESTUDO :

Relato de caso

RELATO DE CASO :

O DN A fo i extraído de sang ue de câmara cardíaca e 1 5 lo ci (D1 S8 0 , Apo B, D1 7 S3 0 , D3 S1 7 4 4 , D1 8 S8 4 9 , D1 2 S1 0 9 0 , FG A, D7 S8 2 0 , D1 S5 3 3 , D9 S3 0 4 , HUMC SF1 PO , HUM TPO X, HUM THO 1 , a me lo g e nina e HLA -DQ A 1 ) fo ra m analisado s utiliz ando -se a técnica da PCR. O s resultado s de to do s o s lo ci analisado s a partir de DN A g enô mico extraído do co rpo carbo niz ado fo ram co mparado s co m o s resultado s o btido s a partir de DN A g enô mico extraído de sang ue do s supo sto s pais bio ló g ico s do co rpo , revelando uma co mpatibilidade g enética.

PA LAV RA S- CH AV E:

C a rb o niz a d o s. Tip a g e m d e DN A . PC R. Identificação humana. Investig ação de paternidade

José Arnaldo Soare s-Vie ira, PhD. Department o f Legal Medicine, Medicine Faculty, Universidade de São Paulo , São Paulo , Brazil.

Ana Elisa Corre ia Bille rbe ck, PhD. Labo rató rio de Ho rmô nio s e Genética Mo lecular, Ho spital das Clínicas da Faculdade de Medicina da Universidade de São Paulo , São Paulo , Brazil.

Edna Sadayo Miazato Iwamura, MSc. Department o f Legal Medicine, Medicine Faculty, Universidade de São Paulo , São Paulo , Brazil.

Laís de Alme ida Cardoso. Department o f Legal Medicine, Medicine Faculty, Universidade de São Paulo , São Paulo , Brazil.

Danie l Rome ro Muñoz, MD, PhD. Department o f Legal Medicine, Medicine Faculty, Universidade de São Paulo , São Paulo , Brazil.

Source s of funding: suppo rted by LIM-HC-FMUSP, Brazil.

Conflict of inte re st: No t declared

Last re ce ive d: 23 Octo ber 1999

Acce pte d: 25 Octo ber 1999

Addre ss for corre sponde nce :

Jo sé Arnaldo So ares-Vieira

Faculdade de Medicina da Universidade de São Paulo - Departamento de Medicina Legal

Rua Teo do ro Sampaio , 115 São Paulo /SP - Brasil - CEP 05405-000